Antisense antiviral compound and method for treating influenza viral infection

ABSTRACT

The invention provides antisense antiviral compounds and methods of their use and production in inhibition of growth of viruses of the Orthomyxoviridae family and in the treatment of a viral infection. The compounds are particularly useful in the treatment of influenza virus infection in a mammal. The antisense antiviral compounds are substantially uncharged, including partially positively charged, morpholino oligonucleotides having 1) a nuclease resistant backbone, 2) 12-40 nucleotide bases, and 3) a targeting sequence of at least 12 bases in length that hybridizes to a target region selected from the following: a) the 5′ or 3′ terminal 25 bases of the negative sense viral RNA segment of Influenzavirus A, Influenzavirus B and Influenzavirus C; b) the terminal 25 bases of the 3′ terminus of the positive sense cRNA and; and c) the 50 bases surrounding the AUG start codon of an influenza viral mRNA.

This is a continuation-in-part of U.S. patent application Ser. No.11/259,434, filed Oct. 25, 2005, which claims the benefit of priority toU.S. Provisional Application No. 60/622,077, filed Oct. 26, 2004. Bothapplications are incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to antisense oligonucleotide compounds for use intreating an influenza virus infection and antiviral treatment methodsemploying the compounds.

REFERENCES

-   Agrawal, S., S. H. Mayrand, et al. (1990). “Site-specific excision    from RNA by RNase H and mixed-phosphate-backbone    oligodeoxynucleotides.” Proc Natl Acad Sci USA 87(4): 1401-5.-   Blommers, M. J., U. Pieles, et al. (1994). “An approach to the    structure determination of nucleic acid analogues hybridized to RNA.    NMR studies of a duplex between 2′-OMe RNA and an oligonucleotide    containing a single amide backbone modification.” Nucleic Acids Res    22(20): 4187-94.-   Bonham, M. A., S. Brown, et al. (1995). “An assessment of the    antisense properties of RNase H-competent and steric-blocking    oligomers.” Nucleic Acids Res 23(7): 1197-203.-   Boudvillain, M., M. Guerin, et al. (1997). “Transplatin-modified    oligo(2′-O-methyl ribonucleotide)s: a new tool for selective    modulation of gene expression.” Biochemistry 36(10): 2925-31.-   Cox, N. J. and K. Subbarao (1999). “Influenza.” Lancet 354(9186):    1277-82.-   Cox, N. J. and K. Subbarao (2000). “Global epidemiology of    influenza: past and present.” Annu Rev Med 51: 407-21.-   Cross, C. W., J. S. Rice, et al. (1997). “Solution structure of an    RNA×DNA hybrid duplex containing a 3′-thioformacetal linker and an    RNA A-tract.” Biochemistry 36(14): 4096-107.-   Dagle, J. M., J. L. Littig, et al. (2000). “Targeted elimination of    zygotic messages in Xenopus laevis embryos by modified    oligonucleotides possessing terminal cationic linkages.” Nucleic    Acids Res 28(10): 2153-7.-   Ding, D., S. M. Grayaznov, et al. (1996). “An    oligodeoxyribonucleotide N3′->P5′ phosphoramidate duplex forms an    A-type helix in solution.” Nucleic Acids Res 24(2): 354-60.-   Egholm, M., O. Buchardt, et al. (1993). “PNA hybridizes to    complementary oligonucleotides obeying the Watson-Crick    hydrogen-bonding rules.” Nature 365(6446): 566-8.-   Felgner, P. L., T. R. Gadek, et al. (1987). “Lipofection: a highly    efficient, lipid-mediated DNA-transfection procedure.” Proc Natl    Acad Sci USA 84(21): 7413-7.-   Gait, M. J., A. S. Jones, et al. (1974). “Synthetic-analogues of    polynucleotides XII. Synthesis of thymidine derivatives containing    an oxyacetamido- or an oxyformamido-linkage instead of a    phosphodiester group.” J Chem Soc [Perkin 1] 0(14): 1684-6.-   Gee, J. E., I. Robbins, et al. (1998). “Assessment of high-affinity    hybridization, RNase H cleavage, and covalent linkage in translation    arrest by antisense oligonucleotides.” Antisense Nucleic Acid Drug    Dev 8(2): 103-11.-   Lesnikowski, Z. J., M. Jaworska, et al. (1990). “Octa(thymidine    methanephosphonates) of partially defined stereochemistry: synthesis    and effect of chirality at phosphorus on binding to    pentadecadeoxyriboadenylic acid.” Nucleic Acids Res 18(8): 2109-15.-   Mertes, M. P. and E. A. Coats (1969). “Synthesis of carbonate    analogs of dinucleosides. 3′-Thymidinyl 5′-thymidinyl carbonate,    3′-thymidinyl 5′-(5-fluoro-2′-deoxyuridinyl) carbonate, and    3′-(5-fluoro-2′-deoxyuridinyl) 5′-thymidinyl carbonate.” J Med Chem    12(1): 154-7.-   Moulton, H. M., M. H. Nelson, et al. (2004). “Cellular uptake of    antisense morpholino oligomers conjugated to arginine-rich    peptides.” Bioconjug Chem 15(2): 290-9.-   Nelson, M. H., D. A. Stein, et al. (2005). “Arginine-rich peptide    conjugation to morpholino oligomers: effects on antisense activity    and specificity.” Bioconjug Chem 16(4): 959-66.-   Strauss, J. H. and E. G. Strauss (2002). Viruses and Human Disease.    San Diego, Academic Press.-   Summerton, J. and D. Weller (1997). “Morpholino antisense oligomers:    design, preparation, and properties.” Antisense Nucleic Acid Drug    Dev 7(3): 187-95.-   Toulme, J. J., R. L. Tinevez, et al. (1996). “Targeting RNA    structures by antisense oligonucleotides.” Biochimie 78(7): 663-73.-   Williams, A. S., J. P. Camilleri, et al. (1996). “A single    intra-articular injection of liposomally conjugated methotrexate    suppresses joint inflammation in rat antigen-induced arthritis.” Br    J Rheumatol 35(8): 719-24.-   Wu, G. Y. and C. H. Wu (1987). “Receptor-mediated in vitro gene    transformation by a soluble DNA carrier system.” J Biol Chem    262(10): 4429-32.

BACKGROUND OF THE INVENTION

Influenza viruses have been a major cause of human mortality andmorbidity throughout recorded history. Influenza A virus infectioncauses millions of cases of severe illness and as many as 500,000 deathseach year worldwide. Epidemics vary widely in severity but occur atregular intervals and always cause significant mortality and morbidity,most frequently in the elderly population. Although vaccines againstmatched influenza strains can prevent illness in 60-80% of healthyadults, the rate of protection is much lower in high-risk groups.Furthermore, vaccination does not provide protection against unexpectedstrains, such as the H5 and H7 avian influenza outbreaks in Hong Kong in1997 and Europe and Southeast Asia in 2003 and 2004. Currentanti-influenza drugs are limited in their capacity to provide protectionand therapeutic effect (Cox and Subbarao 1999; Cox and Subbarao 2000).

Influenza A is a segmented RNA virus of negative-polarity. Genomesegments are replicated by a complex of 4 proteins: the 3 polymerasepolypeptides (PA, PB1 and PB2) and NP (Nucleoprotein). The 5′ and 3′terminal sequence regions of all 8 genome segments are highly conservedwithin a genotype (Strauss and Strauss 2002).

Influenza A viruses can be subtyped according to the antigenic andgenetic nature of their surface glycoproteins; 15 hemagglutinin (HA) and9 neuraminidase (NA) subtypes have been identified to date. Virusesbearing all known HA and NA subtypes have been isolated from avianhosts, but only viruses of the H1N1 (1918), H2N2 (1957/58), and H3N2(1968) subtypes have been associated with widespread epidemics in humans(Strauss and Strauss 2002).

Since 1997, when H5N1 influenza virus was transmitted to humans andkilled 6 of 18 infected persons, there have been multiple transmissionsof avian influenza viruses to mammals. Either the whole virus istransmitted directly or gene segments from the avian influenza virus areacquired by mammalian strains. Widespread infections of poultry withH5N1 viruses in Asia have caused increasing concern that this subtypemay achieve human-to-human spread and establish interspeciestransmission. The species which different types of influenza viruses areable to infect are determined by different forms of the virusglycoproteins (HA, NA). This provides a considerable species barrierbetween birds and humans which is not easily overcome. Pigs, however,provide a “mixing pot”-able to be infected by both types of virus andthereby allowing the passage of avian viruses to humans. When anindividual pig cell is co-infected with both avian and human influenzaviruses, recombinant forms can emerge that carry an avian HA genotypebut readily infect humans. Avian HA can infect pigs, but not humans. Inpigs, during genome segment packaging, it is possible to create a viruswith several Avian segments and Human HA and/or NA segments (Cox andSubbarao 2000).

Influenza viruses infect humans and animals (e.g., pigs, birds, horses)and may cause acute respiratory disease. There have been numerousattempts to produce vaccines effective against influenza virus. None,however, have been completely successful, particularly on a long-termbasis. This may be due, at least in part, to the segmentedcharacteristic of the influenza virus genome, which makes it possible,through re-assortment of the segments, for numerous forms to exist. Forexample, it has been suggested that there could be an interchange of RNAsegments between animal and human influenza viruses, which would resultin the introduction of new antigenic subtypes into both populations.Thus, a long-term vaccination approach has failed, due to the emergenceof new subtypes (antigenic “shift”). In addition, the surface proteinsof the virus, hemagglutinin and neuraminidase, constantly undergo minorantigenic changes (antigenic “drift”). This high degree of variationexplains why specific immunity developed against a particular influenzavirus does not establish protection against new variants. Hence,alternative antiviral strategies are needed. Although influenza B and Cviruses cause less clinical disease than the A types, new antiviraldrugs should also be helpful in curbing infections caused by theseagents.

Influenza viruses that occur naturally among birds are called avianinfluenza (bird flu). The birds carry the viruses in their intestinesbut do not generally get sick from the infection. However, migratorybirds can carry the bird flu to infect domestic chickens, ducks andturkeys causing illness and even death. Avian flu does not easily infecthumans but when human exposure is more frequent, such as contact withdomestic birds, human infections occur. A dangerous bird flu (H5N1) wasfirst identified in terns in South Africa in 1961 and was identified asa potentially deadly form of flu. Outbreaks of H5N1 occurred in eightAsian countries in late 2003 and 2004. At that time more than 100million birds in these countries either died or were killed in order tocontrol the outbreak. Beginning in June of 2004 new deadly outbreaks ofH5N1 were reported in Asia which is currently ongoing. Human infectionsof H5N1 have been observed in Thailand, Vietnam and Cambodia with adeath rate of about 50 percent. These infections have mostly occurredfrom human contact with infected poultry but a few cases ofhuman-to-human spread of H5N1 have occurred.

Currently, there is no vaccine to protect humans against H5N1 butresearch efforts are underway. There are four currently approvedinfluenza medications, amantadine, rimantadine, oseltamivir andzanamivir. Unfortunately, the H5N1 virus is resistant to both amantadineand rimantidine. The remaining oseltamivir and zanamivir may show someefficacy to H5N1 but need to be evaluated more extensively.

In view of the severity of the diseases caused by influenza virusesthere is an immediate need for new therapies to treat influenzainfection. Given the lack of effective prevention or therapies, it istherefore an object of the present invention to provide therapeuticcompounds and methods for treating a host infected with an influenzavirus.

SUMMARY OF THE INVENTION

The invention includes, in one aspect, an anti-viral compound effectivein inhibiting replication within a host cell of an RNA virus having asingle-stranded, negative sense genome and selected from theOrthomyxoviridae family including the Influenzavirus A, Influenzavirus Band Influenzavirus C genera. The compound targets viral RNA sequenceswithin a region selected from the following: 1) the 5′ or 3′ terminal 25bases of the negative sense viral RNA segments; 2) the terminal 25 basesof the 3′ terminus of the positive sense cRNA and; 3) 50 basessurrounding the AUG start codons of influenza viral mRNAs.

The antiviral compound consists of an oligonucleotide analogcharacterized by: a) a nuclease-resistant backbone, b) 12-40 nucleotidebases, and c) a targeting sequence of at least 12 bases in length, thathybridizes to a target region selected from the following: i) the 5′ or3′ terminal 25 bases of a negative sense viral RNA segment ofInfluenzavirus A, Influenzavirus B and Influenzavirus C, ii) theterminal 30 bases of the 3′ terminus of a positive sense cRNA ofInfluenzavirus A, Influenzavirus B and Influenzavirus C, and iii) the 50bases surrounding the AUG start codon of an influenza viral mRNA.

The oligonucleotide analog also has: a) the capability of being activelytaken up by mammalian host cells, and b) the ability to form aheteroduplex structure with the viral target region, wherein saidheteroduplex structure is: i) composed of the positive or negative sensestrand of the virus and the oligonucleotide compound, and ii)characterized by a Tm of dissociation of at least 45° C.

The invention includes, in another aspect, an antiviral compound thatinhibits, in a mammalian host cell, replication of an infectinginfluenza virus having a single-stranded, segmented, negative-sensegenome and selected from the Orthomyxoviridae family. The compound isadministered to the infected host cells as an oligonucleotide analogcharacterized by the elements described above on pp. 5-6. The compoundmay be administered to a mammalian subject infected with the influenzavirus, or at risk of infection with the influenza virus.

The compound may be composed of morpholino subunits linked by uncharged,phosphorus-containing intersubunit linkages, joining a morpholinonitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit.In one embodiment, the intersubunit linkages are phosphorodiamidatelinkages, such as those having the structure:

where Y₁═O, Z=O, Pj is a purine or pyrimidine base-pairing moietyeffective to bind, by base-specific hydrogen bonding, to a base in apolynucleotide, and X is alkyl, alkoxy, thioalkoxy, or alkyl amino,e.g., wherein X═NR₂, where each R is independently hydrogen or methyl.

The compound may be composed of morpholino subunits linked with theuncharged linkages described above interspersed with linkages that arepositively charged at physiological pH. The total number of positivelycharged linkages is between 2 and no more than half of the total numberof linkages. The positively charged linkages have the structure above,where X is 1-piperazine.

The compound may be a covalent conjugate of an oligonucleotide analogmoiety capable of forming such a heteroduplex structure with thepositive or negative sense strand of the virus, and an arginine-richpolypeptide effective to enhance the uptake of the compound into hostcells. Exemplary polypeptides have one of the sequences identified asSEQ ID NOs:25-30.

In a related aspect, the invention includes a heteroduplex complexformed between:

-   -   (a) the 5′ or 3′ terminal 25 bases of the negative sense viral        RNA and/or;    -   (b) the terminal 25 bases of the 3′ terminus of the positive        sense mRNA and/or;    -   (c) 50 bases surrounding the AUG start codons of viral mRNA of        an influenza virus selected from the Orthomyxoviridae family        and,    -   (d) an oligonucleotide analog compound characterized by:        -   (i) a nuclease-resistant backbone,        -   (ii) capable of uptake by mammalian host cells,        -   (iii) containing between 12-40 nucleotide bases,

where said heteroduplex complex has a Tm of dissociation of at least 45°C. and disruption of a stem-loop secondary structure.

An exemplary oligonucleotide analog is composed of morpholino subunitslinked by uncharged, phosphorus-containing intersubunit linkages,joining a morpholino nitrogen of one subunit to a 5′ exocyclic carbon ofan adjacent subunit. The compound may have phosphorodiamidate linkages,such as in the structure

where Y₁═O, Z=O, Pj is a purine or pyrimidine base-pairing moietyeffective to bind, by base-specific hydrogen bonding, to a base in apolynucleotide, and X is alkyl, alkoxy, thioalkoxy, or alkyl amino. In apreferred compound, X═NR₂, where each R is independently hydrogen ormethyl. The compound may also be composed of morpholino subunits linkedwith the uncharged linkages described above interspersed with linkagesthat are positively charged at physiological pH. The total number ofpositively charged linkages is between 2 and no more than half of thetotal number of linkages. The positively charged linkages have thestructure above, where X is 1-piperazine. The compound may be theoligonucleotide analog alone or a conjugate of the analog and anarginine-rich polypeptide capable of enhancing the uptake of thecompound into host cells. Exemplary polypeptides have one of thesequences identified as SEQ ID NOs:25-30.

In still another aspect, the invention includes an oligonucleotideanalog compound for use in inhibiting replication in mammalian hostcells of an influenza virus having a single-stranded, segmented,negative-sense RNA genome and selected from the Orthomyxoviridae family.The compound is characterized by the elements described above on pp.5-6.

An exemplary oligonucleotide analog is composed of morpholino subunitslinked by uncharged, phosphorus-containing intersubunit linkages,joining a morpholino nitrogen of one subunit to a 5′ exocyclic carbon ofan adjacent subunit. The compound may have phosphorodiamidate linkages,such as in the structure

where Y₁═O, Z=O, Pj is a purine or pyrimidine base-pairing moietyeffective to bind, by base-specific hydrogen bonding, to a base in apolynucleotide, and X is alkyl, alkoxy, thioalkoxy, or alkyl amino. In apreferred compound, X═NR₂, where each R is independently hydrogen ormethyl. The compound may be composed of morpholino subunits linked withthe uncharged linkages described above interspersed with linkages thatare positively charged at physiological pH. The total number ofpositively charged linkages is between 2 and no more than half of thetotal number of linkages. The positively charged linkages have thestructure above, where X is 1-piperazine.

The compound may be the oligonucleotide analog alone or a conjugate ofthe analog and an arginine-rich polypeptide capable of enhancing theuptake of the compound into host cells. Exemplary polypeptides have oneof the sequences identified as SEQ ID NOs:25-30.

For treatment of Influenza A virus as given below, the targetingsequence hybridizes to a region associated with one of the group ofsequences identified as SEQ ID NOs:1-9. Preferred targeting sequencesare those complementary to either the minus strand target of SEQ ID NO:4or the positive-strand target of SEQ ID NO:3. Exemplary antisensephosphorodiamidate morpholino oligomers (“PMOs”) that target these tworegions are listed as SEQ ID NOs:12 and 13, respectively.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1D show the repeating subunit segment of several preferredmorpholino oligonucleotides, designated A through D, constructed usingsubunits having 5-atom (A), six-atom (B) and seven-atom (C-D) linkinggroups suitable for forming polymers.

FIGS. 2A-2G show examples of uncharged linkage types in oligonucleotideanalogs. FIG. 2H shows an example of a preferred cationic linkage group.

FIG. 3 shows the three different species of influenza virus RNA presentin infected cells, vRNA, mRNA and cRNA, and the target location oftargeting PMO described herein.

FIG. 4 shows the conservation of target sequences in two importantstereotypes of influenza, H1N1 and H5N1, for each base of two preferredPMOs (PB1-AUG and NP-3′ terminus; SEQ ID NOs:13 and 12). The percentageof isolates having the indicated base is the subscript number after eachbase.

FIGS. 5A-5B show the effect of 20 mM AUG-targeted and termini-targetedPMO on influenza virus replication in infected Vero cells. FIG. 5Cdescribes the experimental protocol.

FIGS. 6A-6C show the dose response of AUG-targeted PMO on influenzavirus replication in Vero cells using the hemagglutinin assay (6A) andthe plaque assay techniques (6B). FIG. 6C describes the experimentalprotocol.

FIGS. 7A-7B show the dose response of termini-targeted PMO on influenzavirus replication in Vero cells using the same assays as in FIG. 6. FIG.7C describes the experimental protocol.

FIGS. 8A-8C show the suppression of transcription of vRNA to mRNA andcRNA by PMOs that target the 3′ terminus of NP vRNA. FIG. 8D describesthe experimental protocol.

FIG. 9A shows the synergistic effect of PMO that target the termini ofthe NP segment and the NP gene AUG start codon on influenza A virusreplication in Vero cells. FIG. 9B describes the experimental protocol.

FIG. 10 shows the synthetic steps to produce subunits used to produce+PMO containing the (1-piperazino) phosphinylideneoxy cationic linkageas shown in FIG. 2H.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

The terms below, as used herein, have the following meanings, unlessindicated otherwise:

“Alkyl” refers to a fully saturated monovalent radical containing carbonand hydrogen, which may be branched, linear, or cyclic (cycloalkyl).Examples of alkyl groups are methyl, ethyl, n-butyl, t-butyl, n-heptyl,isopropyl, cyclopropyl, cyclopentyl, ethylcyclopentyl, and cyclohexyl.Generally preferred are alkyl groups having one to six carbon atoms,referred to as “lower alkyl”, and exemplified by methyl, ethyl, n-butyl,i-butyl, t-butyl, isoamyl, n-pentyl, and isopentyl. In one embodiment,lower alkyl refers to C₁ to C₄ alkyl.

“Alkenyl” refers to an unsaturated monovalent radical containing carbonand hydrogen, which may be branched, linear, or cyclic. The alkenylgroup may be monounsaturated or polyunsaturated. Generally preferred arealkenyl groups having one to six carbon atoms, referred to as “loweralkenyl”.

“Aryl” refers to a substituted or unsubstituted monovalent aromaticradical, generally having a single ring (e.g., benzene) or two condensedrings (e.g., naphthyl). This term includes heteroaryl groups, which arearomatic ring groups having one or more nitrogen, oxygen, or sulfuratoms in the ring, such as furyl, pyrrole, pyridyl, and indole. By“substituted” is meant that one or more ring hydrogens in the aryl groupis replaced with a halide such as fluorine, chlorine, or bromine; with alower alkyl group containing one or two carbon atoms; nitro, amino,methylamino, dimethylamino, methoxy, halomethoxy, halomethyl, orhaloethyl. Preferred substituents include halogen, methyl, ethyl, andmethoxy. Generally preferred are aryl groups having a single ring.

“Aralkyl” refers to an alkyl, preferably lower (C₁-C₄, more preferablyC₁-C₂) alkyl, substituent which is further substituted with an arylgroup; examples are benzyl (—CH₂C₆H₅) and phenethyl (—CH₂CH₂C₆H₅).

“Heterocycle” refers to a non-aromatic ring, preferably a 5- to7-membered ring, whose ring atoms are selected from the group consistingof carbon, nitrogen, oxygen and sulfur. Preferably, the ring atomsinclude 3 to 6 carbon atoms. Such heterocycles include, for example,pyrrolidine, piperidine, piperazine, and morpholine.

The term “substituted”, with respect to an alkyl, alkenyl, alkynyl,aryl, aralkyl, or alkaryl group, refers to replacement of a hydrogenatom with a heteroatom-containing substituent, such as, for example,halogen, hydroxy, alkoxy, thiol, alkylthio, amino, alkylamino, imino,oxo (keto), nitro, cyano, or various acids or esters such as carboxylic,sulfonic, or phosphonic.

The terms “oligonucleotide analog” refers to an oligonucleotide having(i) a modified backbone structure, e.g., a backbone other than thestandard phosphodiester linkage found in natural oligo- andpolynucleotides, and (ii) optionally, modified sugar moieties, e.g.,morpholino moieties rather than ribose or deoxyribose moieties. Theanalog supports bases capable of hydrogen bonding by Watson-Crick basepairing to standard polynucleotide bases, where the analog backbonepresents the bases in a manner to permit such hydrogen bonding in asequence-specific fashion between the oligonucleotide analog moleculeand bases in a standard polynucleotide (e.g., single-stranded RNA orsingle-stranded DNA). Preferred analogs are those having a substantiallyuncharged, phosphorus containing backbone.

A substantially uncharged, phosphorus containing backbone in anoligonucleotide analog is one in which a majority of the subunitlinkages, e.g., between 50-100%, are uncharged at physiological pH, andcontain a single phosphorous atom. The analog contains between 8 and 40subunits, typically about 8-25 subunits, and preferably about 12 to 25subunits. The analog may have exact sequence complementarity to thetarget sequence or near complementarity, as defined below.

A “subunit” of an oligonucleotide analog refers to one nucleotide (ornucleotide analog) unit of the analog. The term may refer to thenucleotide unit with or without the attached intersubunit linkage,although, when referring to a “charged subunit”, the charge typicallyresides within the intersubunit linkage (e.g. a phosphate orphosphorothioate linkage).

A “morpholino oligonucleotide analog” is an oligonucleotide analogcomposed of morpholino subunit structures of the form shown in FIGS.1A-1D, where (i) the structures are linked together byphosphorus-containing linkages, one to three atoms long, joining themorpholino nitrogen of one subunit to the 5′ exocyclic carbon of anadjacent subunit, and (ii) P_(i) and P_(j) are purine or pyrimidinebase-pairing moieties effective to bind, by base-specific hydrogenbonding, to a base in a polynucleotide. The purine or pyrimidinebase-pairing moiety is typically adenine, cytosine, guanine, uracil orthymine. The synthesis, structures, and binding characteristics ofmorpholino oligomers are detailed in U.S. Pat. Nos. 5,698,685,5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,521,063, and 5,506,337,all of which are incorporated herein by reference.

The subunit and linkage shown in FIG. 1B are used for six-atomrepeating-unit backbones, as shown in FIG. 1B (where the six atomsinclude: a morpholino nitrogen, the connected phosphorus atom, the atom(usually oxygen) linking the phosphorus atom to the 5′ exocyclic carbon,the 5′ exocyclic carbon, and two carbon atoms of the next morpholinoring). In these structures, the atom Y₁ linking the 5′ exocyclicmorpholino carbon to the phosphorus group may be sulfur, nitrogen,carbon or, preferably, oxygen. The X moiety pendant from the phosphorusis any stable group which does not interfere with base-specific hydrogenbonding. Preferred X groups include fluoro, alkyl, alkoxy, thioalkoxy,and alkyl amino, including cyclic amines, all of which can be variouslysubstituted, as long as base-specific bonding is not disrupted. Alkyl,alkoxy and thioalkoxy preferably include 1-6 carbon atoms. Alkyl aminopreferably refers to lower alkyl (C₁ to C₆) substitution, and cyclicamines are preferably 5- to 7-membered nitrogen heterocycles optionallycontaining 1-2 additional heteroatoms selected from oxygen, nitrogen,and sulfur. Z is sulfur or oxygen, and is preferably oxygen.

A preferred morpholino oligomer is a phosphorodiamidate-linkedmorpholino oligomer, referred to herein as a PMO. Such oligomers arecomposed of morpholino subunit structures such as shown in FIG. 2B,where X═NH2, NHR, or NR2 (where R is lower alkyl, preferably methyl),Y═O, and Z=O, and Pi and Pj are purine or pyrimidine base-pairingmoieties effective to bind, by base-specific hydrogen bonding, to a basein a polynucleotide, as seen in FIG. 2G. Also preferred are morpholinooligomers where the phosphordiamidate linkages are uncharged linkages asshown in FIG. 2G interspersed with cationic linkages as shown in FIG. 2Hwhere, in FIG. 2B, X=1-piperazino. In another FIG. 2B embodiment,X=lower alkoxy, such as methoxy or ethoxy, Y═NH or NR, where R is loweralkyl, and Z=O The term “substituted”, particularly with respect to analkyl, alkoxy, thioalkoxy, or alkylamino group, refers to replacement ofa hydrogen atom on carbon with a heteroatom-containing substituent, suchas, for example, halogen, hydroxy, alkoxy, thiol, alkylthio, amino,alkylamino, imino, oxo (keto), nitro, cyano, or various acids or esterssuch as carboxylic, sulfonic, or phosphonic. It may also refer toreplacement of a hydrogen atom on a heteroatom (such as an aminehydrogen) with an alkyl, carbonyl or other carbon containing group.

As used herein, the term “target”, relative to the viral genomic RNA,refers to a viral genomic RNA, and specifically, to a region associatedwith stem-loop secondary structure within the 5′-terminal end 40 basesof the positive-sense RNA strand of a single-stranded RNA (ssRNA) virusdescribed herein.

The term “target sequence” refers to a portion of the target RNA againstwhich the oligonucleotide analog is directed, that is, the sequence towhich the oligonucleotide analog will hybridize by Watson-Crick basepairing of a complementary sequence. As will be seen, the targetsequence may be a contiguous region of the viral positive-strand RNA, ormay be composed of complementary fragments of both the 5′ and 3′sequences involved in secondary structure.

The term “targeting sequence” is the sequence in the oligonucleotideanalog that is complementary (meaning, in addition, substantiallycomplementary) to the target sequence in the RNA genome. The entiresequence, or only a portion, of the analog compound may be complementaryto the target sequence. For example, in an analog having 20 bases, only12-14 may be targeting sequences. Typically, the targeting sequence isformed of contiguous bases in the analog, but may alternatively beformed of non-contiguous sequences that when placed together, e.g., fromopposite ends of the analog, constitute sequence that spans the targetsequence. As will be seen, the target and targeting sequences areselected such that binding of the analog is to a region within; 1) the5′ or 3′ terminal 25 bases of the negative sense viral RNA; 2) theterminal 25 bases of the 3′ terminus of the positive sense mRNA and/or;3) 50 bases surrounding the AUG start codons of viral mRNA.

Target and targeting sequences are described as “complementary” to oneanother when hybridization occurs in an antiparallel configuration. Atargeting sequence may have “near” or “substantial” complementarity tothe target sequence and still function for the purpose of the presentinvention, that is, it may still be “complementary.” Preferably, theoligonucleotide analog compounds employed in the present invention haveat most one mismatch with the target sequence out of 10 nucleotides, andpreferably at most one mismatch out of 20. Alternatively, the antisenseoligomers employed have at least 90% sequence homology, and preferablyat least 95% sequence homology, with the exemplary targeting sequencesas designated herein.

An oligonucleotide analog “specifically hybridizes” to a targetpolynucleotide if the oligomer hybridizes to the target underphysiological conditions, with a Tm substantially greater than 45° C.,preferably at least 50° C., and typically 60° C.-80° C. or higher. Suchhybridization preferably corresponds to stringent hybridizationconditions. At a given ionic strength and pH, the Tm is the temperatureat which 50% of a target sequence hybridizes to a complementarypolynucleotide. Again, such hybridization may occur with “near” or“substantial” complementary of the antisense oligomer to the targetsequence, as well as with exact complementarity.

A “nuclease-resistant” oligomeric molecule (oligomer) refers to onewhose backbone is substantially resistant to nuclease cleavage, innon-hybridized or hybridized form; by common extracellular andintracellular nucleases in the body; that is, the oligomer shows littleor no nuclease cleavage under normal nuclease conditions in the body towhich the oligomer is exposed.

A “heteroduplex” refers to a duplex between an oligonculeotide analogand the complementary portion of a target RNA. A “nuclease-resistantheteroduplex” refers to a heteroduplex formed by the binding of anantisense oligomer to its complementary target, such that theheteroduplex is substantially resistant to in vivo degradation byintracellular and extracellular nucleases, such as RNAseH, which arecapable of cutting double-stranded RNA/RNA or RNA/DNA complexes.

A “base-specific intracellular binding event involving a target RNA”refers to the specific binding of an oligonucleotide analog to a targetRNA sequence inside a cell. The base specificity of such binding issequence specific. For example, a single-stranded polynucleotide canspecifically bind to a single-stranded polynucleotide that iscomplementary in sequence.

An “effective amount” of an antisense oligomer, targeted against aninfecting influenza virus, is an amount effective to reduce the rate ofreplication of the infecting virus, and/or viral load, and/or symptomsassociated with the viral infection.

As used herein, the term “body fluid” encompasses a variety of sampletypes obtained from a subject including, urine, saliva, plasma, blood,spinal fluid, or other sample of biological origin, such as skin cellsor dermal debris, and may refer to cells or cell fragments suspendedtherein, or the liquid medium and its solutes.

The term “relative amount” is used where a comparison is made between atest measurement and a control measurement. The relative amount of areagent forming a complex in a reaction is the amount reacting with atest specimen, compared with the amount reacting with a controlspecimen. The control specimen may be run separately in the same assay,or it may be part of the same sample (for example, normal tissuesurrounding a malignant area in a tissue section).

“Treatment” of an individual or a cell is any type of interventionprovided as a means to alter the natural course of the individual orcell. Treatment includes, but is not limited to, administration of e.g.,a pharmaceutical composition, and may be performed eitherprophylactically, or subsequent to the initiation of a pathologic eventor contact with an etiologic agent. The related term “improvedtherapeutic outcome” relative to a patient diagnosed as infected with aparticular virus, refers to a slowing or diminution in the growth ofvirus, or viral load, or detectable symptoms associated with infectionby that particular virus.

An agent is “actively taken up by mammalian cells” when the agent canenter the cell by a mechanism other than passive diffusion across thecell membrane. The agent may be transported, for example, by “activetransport”, referring to transport of agents across a mammalian cellmembrane by e.g. an ATP-dependent transport mechanism, or by“facilitated transport”, referring to transport of antisense agentsacross the cell membrane by a transport mechanism that requires bindingof the agent to a transport protein, which then facilitates passage ofthe bound agent across the membrane. For both active and facilitatedtransport, the oligonucleotide analog preferably has a substantiallyuncharged backbone, as defined below. Alternatively, the antisensecompound may be formulated in a complexed form, such as an agent havingan anionic backbone complexed with cationic lipids or liposomes, whichcan be taken into cells by an endocytotic mechanism. The analog may alsobe conjugated, e.g., at its 5′ or 3′ end, to an arginine-rich peptide,such as a portion of the HIV TAT protein, polyarginine, or tocombinations of arginine and other amino acids including the non-naturalamino acids 6-aminohexanoic acid (Ahx) and beta-alanine (βAla).Exemplary arginine-rich delivery peptides are listed as SEQ IDNOs:25-30. These exemplary arginine-rich delivery peptides facilitatetransport into the target host cell as described (Moulton, Nelson et al.2004; Nelson, Stein et al. 2005).

Rules for the selection of targeting sequences capable of inhibitingreplication of the influenza viral genome are discussed below.

II. TARGETED VIRUSES

The present invention is based on the discovery that effectiveinhibition of single-stranded, segmented, negative-sense RNA viruses canbe achieved by exposing cells infected with influenza virus to antisenseoligonucleotide analog compounds (i) that target 1) the 5′ or 3′terminal 25 bases of the negative sense viral RNA; 2) the terminal 25bases of the 3′ terminus of the positive sense mRNA and/or; 3) 50 basessurrounding the AUG start codons of viral mRNA; and (ii) having physicaland pharmacokinetic features which allow effective interaction betweenthe antisense compound and the virus within host cells. In one aspect,the oligomers can be used in treating a mammalian subject infected withinfluenza virus.

The invention targets RNA viruses having genomes that are: (i) singlestranded, (ii) segmented and (iii) negative polarity. The targetedviruses also synthesize two different versions of a genomic complementof the negative sense virion RNA (vRNA) with positive polarity: 1) cRNAthat is used as a template for replication of negative sense virion RNA,and 2) a complementary positive sense RNA (mRNA) that is used fortranslation of viral proteins. FIG. 3 is a schematic that shows thesedifferent RNA species and the target location of antisense PMO describedin the present invention. In particular, targeted viral families includemembers of the Orthomyxoviridae family including the Influenzavirus A,Influenzavirus B and Influenzavirus C genera. Various physical,morphological, and biological characteristics of members of theOrthomyxoviridae family can be found, for example, in Textbook of HumanVirology, R. Belshe, ed., 2^(nd) Edition, Mosby, 1991, at the UniversalVirus Database of the International Committee on Taxonomy of Viruses(www.ncbi.nlm.nih.gov/ICTVdb/index.htm) and in human virology textbooks(see, for example (Strauss and Strauss 2002). Some of the key biologicalcharacteristics of the Orthomxyoviridae family of viruses are describedbelow.

Influenza Viruses

Influenza A, influenza B and influenza C viruses are the only members ofthe Influenzavirus A, Influenzavirus B and Influenzavirus C genera,respectively. These viruses are membrane-enclosed viruses whose genomesare segmented negative-sense (i.e. minus) strands of RNA ((−)RNA). Theten influenza virus genes are present on eight segments of thesingle-stranded RNA of strains A and B, and on seven segments of strainC. The segments vary in size (from 890 to 2341 nucleotides in length)and each is a template for synthesis of different mRNAs. The influenzavirus virion contains virus-specific RNA polymerases necessary for mRNAsynthesis from these templates and, in the absence of such specificpolymerases, the minus strand of influenza virus RNA is not infectious.Initiation of transcription of the mRNAs occurs when the influenza virusmRNA polymerase takes 12 to 15 nucleotides from the 5′ end of a cellularmRNA or mRNA precursor and uses the borrowed oligonucleotide as aprimer. This process has been termed “cap-snatching” because it places a5′ cap structure on the viral mRNA. Generally, the mRNAs made throughthis process encode only one protein. The M gene and NS gene viral RNAsegments also code for spliced mRNAs, which results in production of twodifferent proteins for each of these two segments.

Replication of influenza viral RNA occurs in the nucleus and involvesthe synthesis of three different species of RNA. A schematic of thisprocess is shown in FIG. 3. After infection of a naïve cell, the minusstrand virion RNA (vRNA) is transported to the nucleus where RNAdestined for translation (mRNA) is synthesized using 5′-terminal 10-13nucleotide primers cleaved by viral-encoded enzymes from capped cellularpre-mRNA molecules (i.e. cap-snatching). Synthesis of each mRNAcontinues to near the end of the genome segment where an oligo(U)stretch is encountered and a poly(A) tail is added. The dedicated viralmRNAs are transported to the cytoplasm for translation and aftersufficient viral proteins are transported back into the nucleus,synthesis of vRNA destined for nascent virions is initiated. An exactantigenomic copy of vRNA is synthesized (termed cRNA) which is a perfectcomplement of the genomic vRNA and serves as a template for productionof new vRNA. The different RNAs synthesized during influenza virusreplication are shown schematically in FIG. 3.

GenBank references for exemplary viral nucleic acid target sequencesrepresenting influenza A genomic segments are listed in Table 1 below.The nucleotide sequence numbers in Table 1 are derived from the Genbankreference for the positive-strand RNA. It will be appreciated that thesesequences are only illustrative of other sequences in theOrthomyxoviridae family, as may be available from availablegene-sequence databases of literature or patent resources. The sequencesbelow, identified as SEQ ID NOs:1-9, are also listed in the SequenceListing at the end of the specification.

The target sequences in Table 1 represent; 1) the 5′ or 3′ terminal 25bases of the negative sense viral RNA (SEQ ID NOs:4-9); 2) the terminal25 bases of the 3′ terminus of the positive sense mRNA (SEQ ID NOs:4-9)and; 3) 50 bases surrounding the AUG start codons of the indicatedinfluenza virus genes (SEQ ID NOs:1-3). The sequences shown are thepositive-strand (i.e., antigenomic or mRNA) sequence in the 5′ to 3′orientation. It will be obvious that when the target is the minus-strandvRNA the targeted sequence is the complement of the sequence listed inTable 1.

Table 1 lists the targets for three different influenza A viral genes,PB2, PB1 and nucleoprotein (NP), encoded by genomic segments 1, 2 and 5,respectively. The PB1, PB2 and NP proteins are components of the viralRNA polymerase and PB2 also functions as the “cap-snatching” enzyme. Thetarget sequences for the AUG start codons of the three genes arerepresented as SEQ ID NOs:1-3. The 3′ terminal sequences of the threegenomic segments are represented by SEQ ID NOs:4, 6 and 8 and can betargeted on both the positive strand and the negative strand of thosesegments. The 5′ terminal sequences (SEQ ID NOs:5, 7 and 9) can besuccessfully targeted on the minus strand. TABLE 1 Exemplary InfluenzaViral Nucleic Acid Target Sequences Nucle- SEQ GenBank otide ID Name No.Region Sequence (5′ to 3′) NO NP-31 J02147  21-UCACUCACUGAGUGACAUCAAAAUCA 1  70 UGGCGUCCCAAGGCACCAAACGGU PB2-11 V00603  1- AGCGAAAGCAGGUCAAUUAUAUUCAA 2  50 UAUGGAAAGAAUAAAAGAACUAAG PB1-J02151   1- AGCGAAAGCAGGCAAACCAUUUGAAU 3 AUG  50GGAUGUCAAUCCGACCUUACUUUU NP- J02147 1541- AAAGAAAAAUACCCUUGUUUCUACU 43′term 1565 NP- J02147   1- AGCAAAAGCAGGGUAGAUAAUCACU 5 5′term  25 PB1-J02151 2317- CAUGAAAAAAUGCCUUGUUCCUACU 6 3′term 2341 PB1- J02151   1-AGCGAAAGCAGGCAAACCAUUUGAA 7 5′term  25 PB2- V00603 2317-GUUUAAAAACGACCUUGUUUCUACU 8 3′term 2341 PB2- V00603   1-AGCGAAAGCAGGUCAAUUAUAUUCA 9 5′term  25

FIG. 4 shows conservation of target sequences in two important serotypesof influenza, H1N1 and H5N1, for each base of two preferred PMOs(PB1-AUG and NP-3′term; SEQ ID NOs:13 and 12) based on Los AlamosNational Laboratory (LANL) influenza database of genome sequences(Macken, C., Lu, H., Goodman, J., & Boykin, L., “The value of a databasein surveillance and vaccine selection,” in Options for the Control ofInfluenza IV. A. D. M. E. Osterhaus, N. Cox & A. W. Hampson (Eds.)Amsterdam: Elsevier Science, 2001, 103-106). The same search wasconducted with the National Library of Medicine GenBank database whichis composed of different sequences for influenza and virtually identicalresults were obtained. The capital letter indicates the PMO base and thesubscript number next to the base indicates the percent conservation forthat base for all the isolates in the database. These data indicate onlybase positions 15 and 16 show any variation for the 3′(−)NP terminus andeven better conservation of sequence in the PB1-AUG target.

Targeting sequences are designed to hybridize to a region of the targetsequence as listed in Table 1. Selected targeting sequences can be madeshorter, e.g., 12 bases, or longer, e.g., 40 bases, and include a smallnumber of mismatches, as long as the sequence is sufficientlycomplementary to disrupt the stem structure(s) upon hybridization withthe target, and forms with the virus positive-strand, a heteroduplexhaving a Tm of 45° C. or greater.

More generally, the degree of complementarity between the target andtargeting sequence is sufficient to form a stable duplex. The region ofcomplementarity of the antisense oligomers with the target RNA sequencemay be as short as 8-11 bases, but is preferably 12-15 bases or more,e.g. 12-20 bases, or 12-25 bases. An antisense oligomer of about 14-15bases is generally long enough to have a unique complementary sequencein the viral genome. In addition, a minimum length of complementarybases may be required to achieve the requisite binding Tm, as discussedbelow.

Oligomers as long as 40 bases may be suitable, where at least a minimumnumber of bases, e.g., 12 bases, are complementary to the targetsequence. In general, however, facilitated or active uptake in cells isoptimized at oligomer lengths less than about 30, preferably less than25. For PMO oligomers, described further below, an optimum balance ofbinding stability and uptake generally occurs at lengths of 15-22 bases.

The oligomer may be 100% complementary to the viral nucleic acid targetsequence, or it may include mismatches, e.g., to accommodate variants,as long as a heteroduplex formed between the oligomer and viral nucleicacid target sequence is sufficiently stable to withstand the action ofcellular nucleases and other modes of degradation which may occur invivo. Oligomer backbones which are less susceptible to cleavage bynucleases are discussed below. Mismatches, if present, are lessdestabilizing toward the end regions of the hybrid duplex than in themiddle. The number of mismatches allowed will depend on the length ofthe oligomer, the percentage of G:C base pairs in the duplex, and theposition of the mismatch(es) in the duplex, according to well understoodprinciples of duplex stability. Although such an antisense oligomer isnot necessarily 100% complementary to the viral nucleic acid targetsequence, it is effective to stably and specifically bind to the targetsequence, such that a biological activity of the nucleic acid target,e.g., expression of viral protein(s), is modulated.

The stability of the duplex formed between the oligomer and the targetsequence is a function of the binding Tm and the susceptibility of theduplex to cellular enzymatic cleavage. The Tm of an antisense compoundwith respect to complementary-sequence RNA may be measured byconventional methods, such as those described by Hames et al., NucleicAcid Hybridization, IRL Press, 1985, pp. 107-108 or as described inMiyada C. G. and Wallace R. B., 1987, Oligonucleotide hybridizationtechniques, Methods Enzymol. Vol. 154 pp. 94-107. Each antisenseoligomer should have a binding Tm, with respect to acomplementary-sequence RNA, of greater than body temperature andpreferably greater than 50° C. Tm's in the range 60-80° C. or greaterare preferred. According to well known principles, the Tm of an oligomercompound, with respect to a complementary-based RNA hybrid, can beincreased by increasing the ratio of C:G paired bases in the duplex,and/or by increasing the length (in base pairs) of the heteroduplex. Atthe same time, for purposes of optimizing cellular uptake, it may beadvantageous to limit the size of the oligomer. For this reason,compounds that show high Tm (50° C. or greater) at a length of 20 basesor less are generally preferred over those requiring greater than 20bases for high Tm values.

The antisense activity of the oligomer may be enhanced by using amixture of uncharged and cationic phosphorodiamidate linkages as shownin FIGS. 2G and 2H. The total number of cationic linkages in theoligomer can vary from 1 to 10, and be interspersed throughout theoligomer. Preferably the number of charged linkages is at least 2 and nomore than half the total backbone linkages, e.g., between 2-8 positivelycharged linkages, and preferably each charged linkages is separatedalong the backbone by at least one, preferably at least two unchargedlinkages. The antisense activity of various oligomers can be measured invitro by fusing the oligomer target region to the 5′ end a reporter gene(e.g. firefly luciferase) and then measuring the inhibition oftranslation of the fusion gene mRNA transcripts in cell free translationassays. The inhibitory properties of oligomers containing a mixture ofuncharged and cationic linkages can be enhanced between, approximately,five to 100 fold in cell free translation assays.

Table 2 below shows exemplary targeting sequences, in a 5′-to-3′orientation, that are complementary to influenza A virus. The sequenceslisted provide a collection of targeting sequences from which targetingsequences may be selected, according to the general class rulesdiscussed above. SEQ ID NOs:10-12, 15, 17, 20, 23 and 24 are antisenseto the positive strand (mRNA or cRNA) of the virus whereas SEQ IDNOs:13, 14, 16, 18, 19, 21 and 22 are antisense to the minus strand(vRNA). Thus, for example, in selecting a target against the 3′ terminusof the minus strand of the NP encoding segment (segment 5 of influenzaA) SEQ ID NOs:13 or 16, or a portion of either sequence effective toblock the function of the 3′ terminus of the minus strand can beselected. TABLE 2 Exemplary Antisense Oligomer Sequences TargetTargeting SEQ. Nucle- GenBank Antisense Oligomer ID PMO otides Acc. No.(5′ to 3′) NO. NP-AUG  39- J02147 CTTGGGACGCCATGATTTTG 10  58 PB2-AUG 24- V00603 CTTTTATTCTTTCCATATTG 11  43 PB1-AUG  13- J02151GACATCCATTCAAATGGTTTG 12  33 (−)NP-   1- J02147 AGCAAAAGCAGGGTAGATAATC13 3′trm  22 (−)NP- 1544- J02147 GAAAAATACCCTTGTTTCTACT 14 5′trm 1565(+)NP- 1544- J02147 AGTAGAAACAAGGGTATTTTTC 15 3′trm 1565 Flu(−)   1-J02147 AGCAAAAGCAGG 16 3′trm  12 Flu(+) 1553- J02147 AGTAGAAACAAGG 173′trm 1565 (−)PB1-   1- J02151 AGCGAAAGCAGGCAAACCAT 18 3′trm  20 (−)PB1-2320- J02151 GAAAAAATGCCTTGTTCCTACT 19 5′trm 2341 (+)PB1- 2320- J02151AGTAGGAACAAGGCATTTTTTC 20 3′trm 2341 (−)PB2-   1- V00603AGCGAAAGCAGGTCAATTAT 21 3′trm  20 (−)PB2- 2320- V00603TAAAAACGACCTTGTTTCTACT 22 5′trm 2341 (+)PB2- 2320- V00603AGTAGAAACAAGGTCGTTTTTA 23 3′trm 2341 (+)NP-   1- J02147AGTCTCGACTTGCTACCTCA 24 5′trm  20

III. ANTISENSE OLIGONUCLEOTIDE ANALOG COMPOUNDS

A. Properties

As detailed above, the antisense oligonucleotide analog compound (theterm “antisense” indicates that the compound is targeted against eitherthe virus' positive-sense strand RNA or negative-sense or minus-strand)has a base sequence targeting a region that includes one or more of thefollowing; 1) the 5′ or 3′ terminal 30 bases of the negative sense viralRNA; 2) the terminal 30 bases of the 3′ terminus of the positive sensemRNA and/or; 3) 50 bases surrounding the AUG start codons of viral mRNA.In addition, the oligomer is able to effectively target infectingviruses, when administered to a host cell, e.g. in an infected mammaliansubject. This requirement is met when the oligomer compound (a) has theability to be actively taken up by mammalian cells, and (b) once takenup, form a duplex with the target RNA with a Tm greater than about 45°C.

As will be described below, the ability to be taken up by cells requiresthat the oligomer backbone be substantially uncharged, and, preferably,that the oligomer structure is recognized as a substrate for active orfacilitated transport across the cell membrane. The ability of theoligomer to form a stable duplex with the target RNA will also depend onthe oligomer backbone, as well as factors noted above, the length anddegree of complementarity of the antisense oligomer with respect to thetarget, the ratio of G:C to A:T base matches, and the positions of anymismatched bases. The ability of the antisense oligomer to resistcellular nucleases promotes survival and ultimate delivery of the agentto the cell cytoplasm.

Below are disclosed methods for testing any given, substantiallyuncharged backbone for its ability to meet these requirements.

B. Active or Facilitated Uptake by Cells

The antisense compound may be taken up by host cells by facilitated oractive transport across the host cell membrane if administered in free(non-complexed) form, or by an endocytotic mechanism if administered incomplexed form.

In the case where the agent is administered in free form, the antisensecompound should be substantially uncharged, meaning that a majority ofits intersubunit linkages are uncharged at physiological pH. Experimentscarried out in support of the invention indicate that a small number ofnet charges, e.g., 1-2 for a 15- to 20-mer oligomer, can in fact enhancecellular uptake of certain oligomers with substantially unchargedbackbones. The charges may be carried on the oligomer itself, e.g., inthe backbone linkages, or may be terminal charged-group appendages.Preferably, the number of charged linkages is no more than one chargedlinkage per four uncharged linkages. More preferably, the number is nomore than one charged linkage per ten, or no more than one per twenty,uncharged linkages. In one embodiment, the oligomer is fully uncharged.

An oligomer may also contain both negatively and positively chargedbackbone linkages, as long as opposing charges are present inapproximately equal number. Preferably, the oligomer does not includeruns of more than 3-5 consecutive subunits of either charge. Forexample, the oligomer may have a given number of anionic linkages, e.g.phosphorothioate or N3′→P5′ phosphoramidate linkages, and a comparablenumber of cationic linkages, such as N,N-diethylenediaminephosphoramidates (Dagle, Littig et al. 2000). The net charge ispreferably neutral or at most 1-2 net charges per oligomer.

In addition to being substantially or fully uncharged, the antisenseagent is preferably a substrate for a membrane transporter system (i.e.a membrane protein or proteins) capable of facilitating transport oractively transporting the oligomer across the cell membrane. Thisfeature may be determined by one of a number of tests for oligomerinteraction or cell uptake, as follows.

A first test assesses binding at cell surface receptors, by examiningthe ability of an oligomer compound to displace or be displaced by aselected charged oligomer, e.g., a phosphorothioate oligomer, on a cellsurface. The cells are incubated with a given quantity of test oligomer,which is typically fluorescently labeled, at a final oligomerconcentration of between about 10-300 nM. Shortly thereafter, e.g.,10-30 minutes (before significant internalization of the test oligomercan occur), the displacing compound is added, in incrementallyincreasing concentrations. If the test compound is able to bind to acell surface receptor, the displacing compound will be observed todisplace the test compound. If the displacing compound is shown toproduce 50% displacement at a concentration of 10× the test compoundconcentration or less, the test compound is considered to bind at thesame recognition site for the cell transport system as the displacingcompound.

A second test measures cell transport, by examining the ability of thetest compound to transport a labeled reporter, e.g., a fluorescencereporter, into cells. The cells are incubated in the presence of labeledtest compound, added at a final concentration between about 10-300 nM.After incubation for 30-120 minutes, the cells are examined, e.g., bymicroscopy, for intracellular label. The presence of significantintracellular label is evidence that the test compound is transported byfacilitated or active transport.

The antisense compound may also be administered in complexed form, wherethe complexing agent is typically a polymer, e.g., a cationic lipid,polypeptide, or non-biological cationic polymer, having an oppositecharge to any net charge on the antisense compound. Methods of formingcomplexes, including bilayer complexes, between anionic oligonucleotidesand cationic lipid or other polymer components, are well known. Forexample, the liposomal composition Lipofectin® (Felgner, Gadek et al.1987), containing the cationic lipid DOTMA(N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride) and theneutral phospholipid DOPE (dioleyl phosphatidyl ethanolamine), is widelyused. After administration, the complex is taken up by cells through anendocytotic mechanism, typically involving particle encapsulation inendosomal bodies.

The antisense compound may also be administered in conjugated form withan arginine-rich peptide linked covalently to the 5′ or 3′ end of theantisense oligomer. The peptide is typically 8-16 amino acids andconsists of a mixture of arginine, and other amino acids includingphenyalanine and cysteine. The use of arginine-rich peptide-PMOconjugates can be used to enhance cellular uptake of the antisenseoligomer (See, e.g. (Moulton, Nelson et al. 2004; Nelson, Stein et al.2005). Exemplary arginine-rich peptides for use in practicing theinvention are listed as SEQ ID NOs:25-30. Non-natural amino acids can beused in combination with naturally occuring amino acids as shown in theSequence listing table for SEQ ID NOs:26-30. In these examples6-aminohexanoic acid (Ahx) and/or beta-alanine (β-Ala) are used.

In some instances, liposomes may be employed to facilitate uptake of theantisense oligonucleotide into cells. (See, e.g., Williams, S. A.,Leukemia 10(12):1980-1989, 1996; Lappalainen et al., Antiviral Res.23:119, 1994; Uhlmann et al., antisense oligonucleotides: a newtherapeutic principle, Chemical Reviews, Volume 90, No. 4, pages544-584, 1990; Gregoriadis, G., Chapter 14, Liposomes, Drug Carriers inBiology and Medicine, pp. 287-341, Academic Press, 1979). Hydrogels mayalso be used as vehicles for antisense oligomer administration, forexample, as described in WO 93/01286. Alternatively, theoligonucleotides may be administered in microspheres or microparticles.(See, e.g. Wu and Wu 1987). Alternatively, the use of gas-filledmicrobubbles complexed with the antisense oligomers can enhance deliveryto target tissues, as described in U.S. Pat. No. 6,245,747.

Alternatively, and according to another aspect of the invention, therequisite properties of oligomers with any given backbone can beconfirmed by a simple in vivo test, in which a labeled compound isadministered to an animal, and a body fluid sample, taken from theanimal several hours after the oligomer is administered, assayed for thepresence of heteroduplex with target RNA. This method is detailed insubsection D below.

C. Substantial Resistance to RNaseH

Two general mechanisms have been proposed to account for inhibition ofexpression by antisense oligonucleotides. (See e.g., Agrawal, Mayrand etal. 1990; Bonham, Brown et al. 1995; Boudvillain, Guerin et al. 1997).In the first, a heteroduplex formed between the oligonucleotide and theviral RNA acts as a substrate for RNaseH, leading to cleavage of theviral RNA. Oligonucleotides belonging, or proposed to belong, to thisclass include phosphorothioates, phosphotriesters, and phosphodiesters(unmodified “natural” oligonucleotides). Such compounds expose the viralRNA in an oligomer:RNA duplex structure to hydrolysis by RNaseH, andtherefore loss of function.

A second class of oligonucleotide analogs, termed “steric blockers” or,alternatively, “RNaseH inactive” or “RNaseH resistant”, have not beenobserved to act as a substrate for RNaseH, and are believed to act bysterically blocking target RNA nucleocytoplasmic transport, splicing ortranslation. This class includes methylphosphonates (Toulme, Tinevez etal. 1996), morpholino oligonucleotides, peptide nucleic acids (PNA's),certain 2′-O-allyl or 2′-O-alkyl modified oligonucleotides (Bonham,Brown et al. 1995), and N3′→P5′ phosphoramidates (Ding, Grayaznov et al.1996; Gee, Robbins et al. 1998).

A test oligomer can be assayed for its RNaseH resistance by forming anRNA:oligomer duplex with the test compound, then incubating the duplexwith RNaseH under a standard assay conditions, as described in Stein etal. After exposure to RNaseH, the presence or absence of intact duplexcan be monitored by gel electrophoresis or mass spectrometry.

D. In Vivo Uptake

In accordance with another aspect of the invention, there is provided asimple, rapid test for confirming that a given antisense oligomer typeprovides the required characteristics noted above, namely, high Tm,ability to be actively taken up by the host cells, and substantialresistance to RNaseH. This method is based on the discovery that aproperly designed antisense compound will form a stable heteroduplexwith the complementary portion of the viral RNA target when administeredto a mammalian subject, and the heteroduplex subsequently appears in theurine (or other body fluid). Details of this method are also given inco-owned U.S. patent application Ser. No. 09/736,920, entitled“Non-Invasive Method for Detecting Target RNA” (Non-Invasive Method),the disclosure of which is incorporated herein by reference.

Briefly, a test oligomer containing a backbone to be evaluated, having abase sequence targeted against a known RNA, is injected into a mammaliansubject. The antisense oligomer may be directed against anyintracellular RNA, including a host RNA or the RNA of an infectingvirus. Several hours (typically 8-72) after administration, the urine isassayed for the presence of the antisense-RNA heteroduplex. Ifheteroduplex is detected, the backbone is suitable for use in theantisense oligomers of the present invention.

The test oligomer may be labeled, e.g. by a fluorescent or a radioactivetag, to facilitate subsequent analyses, if it is appropriate for themammalian subject. The assay can be in any suitable solid-phase or fluidformat. Generally, a solid-phase assay involves first binding theheteroduplex analyte to a solid-phase support, e.g., particles or apolymer or test-strip substrate, and detecting the presence/amount ofheteroduplex bound. In a fluid-phase assay, the analyte sample istypically pretreated to remove interfering sample components. If theoligomer is labeled, the presence of the heteroduplex is confirmed bydetecting the label tags. For non-labeled compounds, the heteroduplexmay be detected by immunoassay if in solid phase format or by massspectroscopy or other known methods if in solution or suspension format.

When the antisense oligomer is complementary to a virus-specific regionof the viral genome (such as those regions of influenza RNA, asdescribed above) the method can be used to detect the presence of agiven influenza virus, or reduction in the amount of virus during atreatment method.

E. Exemplary Oligomer Backbones

Examples of nonionic linkages that may be used in oligonucleotideanalogs are shown in FIGS. 2A-2G. In these figures, B represents apurine or pyrimidine base-pairing moiety effective to bind, bybase-specific hydrogen bonding, to a base in a polynucleotide,preferably selected from adenine, cytosine, guanine and uracil. Suitablebackbone structures include carbonate (3A, R═O) and carbamate (2A,R═NH₂) linkages (Mertes and Coats 1969; Gait, Jones et al. 1974); alkylphosphonate and phosphotriester linkages (2B, R=alkyl or —O-alkyl)(Lesnikowski, Jaworska et al. 1990); amide linkages (2C) (Blommers,Pieles et al. 1994); sulfone and sulfonamide linkages (2D, R₁, R₂═CH₂);and a thioformacetyl linkage (2E) (Cross, Rice et al. 1997). The latteris reported to have enhanced duplex and triplex stability with respectto phosphorothioate antisense compounds (Cross, Rice et al. 1997). Alsoreported are the 3′-methylene-N-methylhydroxyamino compounds ofstructure 2F. Also shown is a cationic linkage in FIG. 2H wherein thenitrogen pendant to the phosphate atom in the linkage of FIG. 2G isreplaced with a 1-piperazino structure. The method for synthesizing the1-piperazino group linkages is described below with respect to FIG. 10.

As noted above, the substantially uncharged oligomer may advantageouslyinclude a limited number of charged backbone linkages. One example of acationic charged phophordiamidate linkage is shown in FIG. 2H. Thislinkage, in which the dimethylamino group shown in FIG. 2G is replacedby a 1-piperazino group as shown in FIG. 2G, can be substituted for anylinkage(s) in the oligomer. By including between two to eight suchcationic linkages, and more generally, at least two and no more thanabout half the total number of linkages, interspersed along the backboneof the otherwise uncharged oligomer, antisense activity can be enhancedwithout a significant loss of specificity. The charged linkages arepreferably separated in the backbone by at least 1 and preferably 2 ormore uncharged linkages.

Peptide nucleic acids (PNAs) are analogs of DNA in which the backbone isstructurally homomorphous with a deoxyribose backbone, consisting ofN-(2-aminoethyl) glycine units to which pyrimidine or purine bases areattached. PNAs containing natural pyrimidine and purine bases hybridizeto complementary oligonucleotides obeying Watson-Crick base-pairingrules, and mimic DNA in terms of base pair recognition (Egholm, Buchardtet al. 1993). The backbone of PNAs is formed by peptide bonds ratherthan phosphodiester bonds, making them well-suited for antisenseapplications. The backbone is uncharged, resulting in PNA/DNA or PNA/RNAduplexes which exhibit greater than normal thermal stability. PNAs arenot recognized by nucleases or proteases.

A preferred oligomer structure employs morpholino-based subunits bearingbase-pairing moieties, joined by uncharged linkages, as described above.Especially preferred is a substantially unchargedphosphorodiamidate-linked morpholino oligomer, such as illustrated inFIGS. 1A-1D, and FIG. 2G. Morpholino oligonucleotides, includingantisense oligomers, are detailed, for example, in co-owned U.S. Pat.Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,185, 444,5,521,063, and 5,506,337, all of which are expressly incorporated byreference herein.

Important properties of the morpholino-based subunits include: theability to be linked in a oligomeric form by stable, uncharged backbonelinkages; the ability to support a nucleotide base (e.g. adenine,cytosine, guanine or uracil) such that the polymer formed can hybridizewith a complementary-base target nucleic acid, including target RNA,with high Tm, even with oligomers as short as 10-14 bases; the abilityof the oligomer to be actively transported into mammalian cells; and theability of the oligomer:RNA heteroduplex to resist RNAse degradation.

Exemplary backbone structures for antisense oligonucleotides of theinvention include the β-morpholino subunit types shown in FIGS. 1A-1D,each linked by an uncharged, phosphorus-containing subunit linkage. FIG.1A shows a phosphorus-containing linkage which forms the five atomrepeating-unit backbone, where the morpholino rings are linked by a1-atom phosphoamide linkage. FIG. 1B shows a linkage which produces a6-atom repeating-unit backbone. In this structure, the atom Y linkingthe 5′ morpholino carbon to the phosphorus group may be sulfur,nitrogen, carbon or, preferably, oxygen. The X moiety pendant from thephosphorus may be fluorine, an alkyl or substituted alkyl, an alkoxy orsubstituted alkoxy, a thioalkoxy or substituted thioalkoxy, orunsubstituted, monosubstituted, or disubstituted nitrogen, includingcyclic structures, such as morpholines or piperidines. Alkyl, alkoxy andthioalkoxy preferably include 1-6 carbon atoms. The Z moieties aresulfur or oxygen, and are preferably oxygen.

The linkages shown in FIGS. 1C and 1D are designed for 7-atomunit-length backbones. In Structure 1C, the X moiety is as in Structure1B, and the Y moiety may be methylene, sulfur, or, preferably, oxygen.In Structure 1D, the X and Y moieties are as in Structure 1B.Particularly preferred morpholino oligonucleotides include thosecomposed of morpholino subunit structures of the form shown in FIG. 1B,where X═NH₂ or N(CH₃)₂, Y═O, and Z=O.

As noted above, the substantially uncharged oligomer may advantageouslyinclude a limited number of charged linkages, e.g. up to about 1 perevery 5 uncharged linkages, more preferably up to about 1 per every 10uncharged linkages. Therefore a small number of charged linkages, e.g.charged phosphoramidate or phosphorothioate, may also be incorporatedinto the oligomers.

The antisense compounds can be prepared by stepwise solid-phasesynthesis, employing methods detailed in the references cited above. Insome cases, it may be desirable to add additional chemical moieties tothe antisense compound, e.g. to enhance pharmacokinetics or tofacilitate capture or detection of the compound. Such a moiety may becovalently attached, typically to a terminus of the oligomer, accordingto standard synthetic methods. For example, addition of apolyethyleneglycol moiety or other hydrophilic polymer, e.g., one having10-100 monomeric subunits, may be useful in enhancing solubility. One ormore charged groups, e.g., anionic charged groups such as an organicacid, may enhance cell uptake. A reporter moiety, such as fluorescein ora radiolabeled group, may be attached for purposes of detection.Alternatively, the reporter label attached to the oligomer may be aligand, such as an antigen or biotin, capable of binding a labeledantibody or streptavidin. In selecting a moiety for attachment ormodification of an antisense oligomer, it is generally of coursedesirable to select chemical compounds of groups that are biocompatibleand likely to be tolerated by a subject without undesirable sideeffects.

IV. INHIBITION OF INFLUENZA VIRAL REPLICATION

The antisense compounds detailed above are useful in inhibitingreplication of single-stranded, negative-sense, segmented RNA viruses ofthe Orthomyxoviridae family. In one embodiment, such inhibition iseffective in treating infection of a host animal by these viruses.Accordingly, the method comprises, in one embodiment, contacting a cellinfected with the virus with a antisense agent effective to inhibit thereplication of the specific virus. In this embodiment, the antisenseagent is administered to a mammalian subject, e.g., human or domesticanimal, infected with a given virus, in a suitable pharmaceuticalcarrier. It is contemplated that the antisense oligonucleotide arreststhe growth of the RNA virus in the host. The RNA virus may be decreasedin number or eliminated with little or no detrimental effect on thenormal growth or development of the host.

In the present invention as described in the Examples,Phosphorodiamidate Morpholino Oligomers (PMOs), designed to hybridize tovarious gene segments of influenza A virus, were evaluated for theirability to inhibit influenza virus production in Vero cell culture. ThePMOs were conjugated to a short arginine-rich peptide to facilitateentry into cells in culture. Vero cells were incubated with PMOcompounds, inoculated with influenza virus, and viral titer determinedby hemagglutinin assay and/or plaque-assay. The compounds targeting theAUG translation start-sites of polymerase component PB1 and nuclearcapsid protein (NP), the 5′ and 3′ ends of vRNA NP segment and the 3′endof cRNA NP segment were very effective, reducing the titer of influenzavirus by 1 to 3 orders of magnitude compared to controls, in adose-dependent and sequence-specific manner over a period of 2 days.Combinations of some of the PMOs exhibited a synergistic antiviraleffect as described in Example 3. These data indicate that several ofthe PMOs tested in this study are potential influenza A therapeutics.

The effective anti-influenza A PMO compounds were observed not to alterthe titer of influenza B virus grown in Vero cells due to the lack ofhomology between the influenza A virus-specific PMOs and thecorresponding influenza B virus targets. However, the PMO describedherein (SEQ ID NOs:10-24) will target most, if not all, influenza Avirus strains because of the high degree of homology between strains atthe respective targets (SEQ ID NOs:1-9). An example of the sequenceconservation at two preferred targets is shown in FIG. 4. In this casethe sequence conservation between multiple isolates of H5N1 (e.g. birdflu) and H1N1 was determined for the targets of the PB1-AUG and (−)NP-3′trm PMOs (SEQ ID NOs:12 and 13, respectively). FIG. 4 shows a veryhigh level of conservation at these target sites.

A. Identification of the Infective Agent

The specific virus causing the infection can be determined by methodsknown in the art, e.g. serological or cultural methods, or by methodsemploying the antisense oligomers of the present invention.

Serological identification employs a viral sample or culture isolatedfrom a biological specimen, e.g., stool, urine, cerebrospinal fluid,blood, etc., of the subject. Immunoassay for the detection of virus isgenerally carried out by methods routinely employed by those of skill inthe art, e.g., ELISA or Western blot. In addition, monoclonal antibodiesspecific to particular viral strains or species are often commerciallyavailable.

Culture methods may be used to isolate and identify particular types ofvirus, by employing techniques including, but not limited to, comparingcharacteristics such as rates of growth and morphology under variousculture conditions.

Another method for identifying the viral infective agent in an infectedsubject employs one or more antisense oligomers targeting broad familiesand/or genera of viruses. Sequences targeting any characteristic viralRNA can be used. The desired target sequences are preferably (i) commonto broad virus families/genera, and (ii) not found in humans.Characteristic nucleic acid sequences for a large number of infectiousviruses are available in public databases, and may serve as the basisfor the design of specific oligomers.

For each plurality of oligomers, the following steps are carried out:(a) the oligomer(s) are administered to the subject; (b) at a selectedtime after said administering, a body fluid sample is obtained from thesubject; and (c) the sample is assayed for the presence of anuclease-resistant heteroduplex comprising the antisense oligomer and acomplementary portion of the viral genome. Steps (a)-(c) are carried forat least one such oligomer, or as many as is necessary to identify thevirus or family of viruses. Oligomers can be administered and assayedsequentially or, more conveniently, concurrently. The virus isidentified based on the presence (or absence) of a heteroduplexcomprising the antisense oligomer and a complementary portion of theviral genome of the given known virus or family of viruses.

Preferably, a first group of oligomers, targeting broad families, isutilized first, followed by selected oligomers complementary to specificgenera and/or species and/or strains within the broad family/genusthereby identified. This second group of oligomers includes targetingsequences directed to specific genera and/or species and/or strainswithin a broad family/genus. Several different second oligomercollections, i.e. one for each broad virus family/genus tested in thefirst stage, are generally provided. Sequences are selected which are(i) specific for the individual genus/species/strains being tested and(ii) not found in humans.

B. Administration of the Antisense Oligomer

Effective delivery of the antisense oligomer to the target nucleic acidis an important aspect of treatment. In accordance with the invention,routes of antisense oligomer delivery include, but are not limited to,various systemic routes, including oral and parenteral routes, e.g.,intravenous, subcutaneous, intraperitoneal, and intramuscular, as wellas inhalation, transdermal and topical delivery. The appropriate routemay be determined by one of skill in the art, as appropriate to thecondition of the subject under treatment. For example, an appropriateroute for delivery of an antisense oligomer in the treatment of a viralinfection of the skin is topical delivery, while delivery of a antisenseoligomer for the treatment of a viral respiratory infection is byinhalation. The oligomer may also be delivered directly to the site ofviral infection, or to the bloodstream.

The antisense oligomer may be administered in any convenient vehiclewhich is physiologically acceptable. Such a composition may include anyof a variety of standard pharmaceutically acceptable carriers employedby those of ordinary skill in the art. Examples include, but are notlimited to, saline, phosphate buffered saline (PBS), water, aqueousethanol, emulsions, such as oil/water emulsions or triglycerideemulsions, tablets and capsules. The choice of suitable physiologicallyacceptable carrier will vary dependent upon the chosen mode ofadministration.

In some instances, liposomes may be employed to facilitate uptake of theantisense oligonucleotide into cells. (See, e.g., Williams, S. A.,Leukemia 10(12):1980-1989, 1996; Lappalainen et al., Antiviral Res.23:119, 1994; Uhlmann et al., antisense oligonucleotides: a newtherapeutic principle, Chemical Reviews, Volume 90, No. 4, pages544-584, 1990; Gregoriadis, G., Chapter 14, Liposomes, Drug Carriers inBiology and Medicine, pp. 287-341, Academic Press, 1979). Hydrogels mayalso be used as vehicles for antisense oligomer administration, forexample, as described in WO 93/01286. Alternatively, theoligonucleotides may be administered in microspheres or microparticles.(See, e.g., Wu, G. Y. and Wu, C. H., J. Biol. Chem. 262:4429-4432,1987). Alternatively, the use of gas-filled microbubbles complexed withthe antisense oligomers can enhance delivery to target tissues, asdescribed in U.S. Pat. No. 6,245,747.

Sustained release compositions may also be used. These may includesemipermeable polymeric matrices in the form of shaped articles such asfilms or microcapsules.

In one aspect of the method, the subject is a human subject, e.g., apatient diagnosed as having a localized or systemic viral infection. Thecondition of a patient may also dictate prophylactic administration ofan antisense oligomer of the invention, e.g. in the case of a patientwho (1) is immunocompromised; (2) is a burn victim; (3) has anindwelling catheter; or (4) is about to undergo or has recentlyundergone surgery. In one preferred embodiment, the oligomer is aphosphorodiamidate morpholino oligomer, contained in a pharmaceuticallyacceptable carrier, and is delivered orally. In another preferredembodiment, the oligomer is a phosphorodiamidate morpholino oligomer,contained in a pharmaceutically acceptable carrier, and is deliveredintravenously (i.v.).

In another application of the method, the subject is a livestock animal,e.g., a chicken, turkey, pig, cow or goat, etc, and the treatment iseither prophylactic or therapeutic. The invention also includes alivestock and poultry food composition containing a food grainsupplemented with a subtherapeutic amount of an antiviral antisensecompound of the type described above. Also contemplated is, in a methodof feeding livestock and poultry with a food grain supplemented withsubtherapeutic levels of an antiviral, an improvement in which the foodgrain is supplemented with a subtherapeutic amount of an antiviraloligonucleotide composition as described above.

The antisense compound is generally administered in an amount and mannereffective to result in a peak blood concentration of at least 200-400 nMantisense oligomer. Typically, one or more doses of antisense oligomerare administered, generally at regular intervals, for a period of aboutone to two weeks. Preferred doses for oral administration are from about1-100 mg oligomer per 70 kg. In some cases, doses of greater than 100 mgoligomer/patient may be necessary. For i.v. administration, preferreddoses are from about 0.5 mg to 100 mg oligomer per 70 kg. The antisenseoligomer may be administered at regular intervals for a short timeperiod, e.g., daily for two weeks or less. However, in some cases theoligomer is administered intermittently over a longer period of time.Administration may be followed by, or concurrent with, administration ofan antibiotic or other therapeutic treatment. The treatment regimen maybe adjusted (dose, frequency, route, etc.) as indicated, based on theresults of immunoassays, other biochemical tests and physiologicalexamination of the subject under treatment.

C. Monitoring of Treatment

An effective in vivo treatment regimen using the antisenseoligonucleotides of the invention may vary according to the duration,dose, frequency and route of administration, as well as the condition ofthe subject under treatment (i.e., prophylactic administration versusadministration in response to localized or systemic infection).Accordingly, such in vivo therapy will often require monitoring by testsappropriate to the particular type of viral infection under treatment,and corresponding adjustments in the dose or treatment regimen, in orderto achieve an optimal therapeutic outcome. Treatment may be monitored,e.g., by general indicators of infection, such as complete blood count(CBC), nucleic acid detection methods, immunodiagnostic tests, viralculture, or detection of heteroduplex.

The efficacy of an in vivo administered antisense oligomer of theinvention in inhibiting or eliminating the growth of one or more typesof RNA virus may be determined from biological samples (tissue, blood,urine etc.) taken from a subject prior to, during and subsequent toadministration of the antisense oligomer. Assays of such samples include(1) monitoring the presence or absence of heteroduplex formation withtarget and non-target sequences, using procedures known to those skilledin the art, e.g., an electrophoretic gel mobility assay; (2) monitoringthe amount of viral protein production, as determined by standardtechniques such as ELISA or Western blotting, or (3) measuring theeffect on viral titer, e.g. by the method of Spearman-Karber. (See, forexample, Pari, G. S. et al., Antimicrob. Agents and Chemotherapy39(5):1157-1161, 1995; Anderson, K. P. et al., Antimicrob. Agents andChemotherapy 40(9):2004-2011, 1996, Cottral, G. E. (ed) in: Manual ofStandard Methods for Veterinary Microbiology, pp. 60-93, 1978).

A preferred method of monitoring the efficacy of the antisense oligomertreatment is by detection of the antisense-RNA heteroduplex. At selectedtime(s) after antisense oligomer administration, a body fluid iscollected for detecting the presence and/or measuring the level ofheteroduplex species in the sample. Typically, the body fluid sample iscollected 3-24 hours after administration, preferably about 6-24 hoursafter administering. As indicated above, the body fluid sample may beurine, saliva, plasma, blood, spinal fluid, or other liquid sample ofbiological origin, and may include cells or cell fragments suspendedtherein, or the liquid medium and its solutes. The amount of samplecollected is typically in the 0.1 to 10 ml range, preferably about 1 mlor less.

The sample may be treated to remove unwanted components and/or to treatthe heteroduplex species in the sample to remove unwanted ssRNA overhangregions, e.g., by treatment with RNase. It is, of course, particularlyimportant to remove overhang where heteroduplex detection relies on sizeseparation, e.g., electrophoresis of mass spectroscopy.

A variety of methods are available for removing unwanted components fromthe sample. For example, since the heteroduplex has a net negativecharge, electrophoretic or ion exchange techniques can be used toseparate the heteroduplex from neutral or positively charged material.The sample may also be contacted with a solid support having asurface-bound antibody or other agent specifically able to bind theheteroduplex. After washing the support to remove unbound material, theheteroduplex can be released in substantially purified form for furtheranalysis, e.g., by electrophoresis, mass spectroscopy or immunoassay.

V. EXAMPLES

The following examples illustrate but are not intended in any way tolimit the invention.

Materials and Methods

Standard recombinant DNA techniques were employed in all constructions,as described in Ausubel, F M et al., in CURRENT PROTOCOLS IN MOLBCULARBIOLOGY, John Wiley and Sons, Inc., Media, Pa., 1992 and Sambrook, J. etal., in MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., Vol. 2, 1989).

All peptides were custom synthesized by Global Peptide Services (Ft.Collins, Colo.) or at AVI BioPharma (Corvallis, Oreg.) and purifiedto >90% purity (see Example 2 below). PMOs were synthesized at AVIBioPharma in accordance with known methods, as described, for example,in ((Summerton and Weller 1997) and U.S. Pat. No. 5,185,444. Thestructure of the PMO is as shown in FIG. 2G.

PMO oligomers were conjugated at the 5′ end with an arginine-richpeptide (R₅F₂R₄C-5′-PMO, SEQ ID NO:25) to enhance cellular uptake asdescribed (U.S. Patent Application 60/466,703 and (Moulton, Nelson etal. 2004; Nelson, Stein et al. 2005).

A schematic of a synthetic pathway that can be used to make morpholinosubunits containing a (1-piperazino) phosphinylideneoxy linkage is shownin FIG. 10; further experimental detail for a representative synthesisis provided in Materials and Methods, below. As shown in the Figure,reaction of piperazine and trityl chloride gave trityl piperazine (1a),which was isolated as the succinate salt. Reaction with ethyltrifluoroacetate (1b) in the presence of a weak base (such asdiisopropylethylamine or DIEA) provided 1-trifluoroacetyl-4-tritylpiperazine (2), which was immediately reacted with HCl to provide thesalt (3) in good yield. Introduction of the dichlorophosphoryl moietywas performed with phosphorus oxychloride in toluene.

The acid chloride (4) is reacted with morpholino subunits (moN), whichmay be prepared as described in U.S. Pat. No. 5,185,444 or in Summertonand Weller, 1997 (cited above), to provide the activated subunits(5,6,7). Suitable protecting groups are used for the nucleoside bases,where necessary; for example, benzoyl for adenine and cytosine,isobutyryl for guanine, and pivaloylmethyl for inosine. The subunitscontaining the (1-piperazino) phosphinylideneoxy linkage can beincorporated into the existing PMO synthesis protocol, as described, forexample in Summerton and Weller (1997), without modification.

Example 1 Inhibition of Influenza A virus in Cell Culture withPhosphorodiamidate Morpholino Oligomers

Phosphorodiamidate Morpholino Oligomers (PMOs), designed to hybridize tovarious gene segments of influenza A virus, were evaluated for theirability to inhibit influenza virus production in Vero cell culture. ThePMOs were conjugated to a short arginine-rich peptide (R₅F₂R₄C) tofacilitate entry into cells in culture. Vero cells were incubated withPMO compounds, inoculated with influenza A virus (strain PR8, H1N1), andviral titer determined by hemagglutinin assay (HA) and/or plaque-assay(CFU). The PMO compounds targeting the AUG translation start-sites ofpolymerase component PB1 and nuclear capsid protein (NP) (SEQ ID NOs: 10and 11), the 5′ and 3′ ends of the NP gene (SEQ ID NOs:13 and 14)encoded by the viral RNA (i.e. vRNA) and the 3′ end of the NP gene (SEQID NO:15) encoded by the complementary RNA (cRNA) were very effective inreducing the titer of influenza virus by 1 to 3 orders of magnitudecompared to controls, in a dose-dependent and sequence-specific mannerover a period of 2 days.

FIG. 5 shows the effect of 20 μM AUG-targeted and 5′ and 3′termini-targeted PMOs on influenza A virus production in Vero cellscompared to untreated (NT) and a scramble control PMO (Scr). Vero cellswere preincubated with PMO for 6 hours followed by virus infection at amultiplicity of infection (M.O.I.) of 0.05. At various timespost-infection, supernatant was collected for determination of virustiter as measured by a Hemaglutinin Assay (HA). Three PMO targeting AUGstart codons of the NP, PB1 and PB2 genes were effective at reducinginfluenza virus replication (SEQ ID NOs:10-12) as shown in FIG. 5. TwoPMOs that target the 5′ and 3′-termini of vRNA, NP(−)5′ and NP(−)3′ (SEQID NOs:13 and 14) and one PMO that targets the 3′ termini of the cRNA,NP(+)3′ (SEQ ID NO:15) were effective at reducing influenza virusreplication in this assay as shown in FIG. 5. The PMO that targets the5′ termini of the cRNA, NP(+)5′ (SEQ ID NO:24), was less effective butstill demonstrated anti-viral activity.

PMOs that target the AUG start codons of three influenza virus genes,NP-AUG, PB2-AUG and PB1-AUG (SEQ ID NOs: 0-12, respectively) wereassayed for their ability to inhibit influenza A virus replication in adose response assay using the hemagglutinin assay and the plaque-formingassay. The results are shown in FIG. 6. For all three PMOs theconcentration that effectively resulted in a 50% reduction in viralreplication (EC50) was found to be less than 1 μM. A three-log reductionin viral replication using the plaque assay was observed for two of thePMOs, NP-AUG and PB1-AUG at 10 μM. Identical assays were performed usingthe termini-targeted PMO: NP(−)3′, NP(−)5′, NP(+)3′ and NP(+)5′ (SEQ IDNOs:13, 14, 15 and 24, respectively). All four PMOs demonstratedsignificant reduction in viral titer as shown in FIG. 7.

Example 2 Effect of PMO Targeting the 3′-Terminus of NP vRNA on NP mRNAand cRNA Transcription

Quantitative RTPCR was used to determine the effect of one of thetermini-targeted PMO, NP(−)3′ (SEQ ID NO:13) on the transcription of theNP vRNA segment into mRNA and cRNA species (i.e., see FIG. 4). The mRNAtranscription product is positive-sense RNA whereas the cRNA is anegative-sense RNA. The NP(−)3′ PMO was incubated with Vero cells for 6hours followed by influenza A virus infection at an MOI of 0.05. Threehours post-infection, RNA was isolated and RNA species specific reversetranscription (RT) was performed followed by quantitative PCR on thereaction product. FIG. 8 shows that the NP(−)3′ PMO (SEQ ID NO:13) thattargets the 3′ end of the vRNA strongly suppressed the transcription ofNP mRNA and cRNA.

Example 3 Synergistic Inhibition of Influenza a Virus Replication inCell Culture Using Combinations of Anti-influenza PMO

Combinations of some of the PMOs exhibited a synergistic antiviraleffect. FIG. 9 shows the synergistic effect of various combinations ofPMO that target the NP vRNA termini and the NP-AUG region. PMO treatmentand influenza A virus infection were as described in Example 1. Theplaque assay was used to measure virus replication. Threetermini-targeted PMO, NP(−)32′, NP(−)5′, NP(+)3′ (SEQ ID NOs:13-15) andthe NP-AUG PMO (SEQ ID NO:10) were mixed in various combinations asshown in FIG. 8. One combination, NP(+)3′ with NP(−)5′ did not produceantiviral activity as this pair of PMO are predicted to hybridize toeach other. All the other PMO combinations demonstrated significantinhibition of influenza A viral replication.

1. An antiviral compound comprising an oligonucleotide analog having a)a nuclease-resistant backbone, b) 12-40 nucleotide bases, and c) atargeting sequence of at least 12 bases in length that hybridizes to atarget region selected from the following: i) the 5′ or 3′ terminal 25bases of a negative sense viral RNA segment of Influenzavirus A,Influenzavirus B and Influenzavirus C, ii) the terminal 25 bases of the3′ terminus of a positive sense cRNA of Influenzavirus A, InfluenzavirusB and Influenzavirus C, and iii) the 50 bases surrounding the AUG startcodon of an influenza viral mRNA, wherein said oligonucleotide analogfurther has: a) the capability of being actively taken up by mammalianhost cells, and b) the ability to form a heteroduplex structure with theviral target region, wherein said heteroduplex structure is: i) composedof the positive or negative sense strand of the virus and theoligonucleotide compound, and ii) characterized by a Tm of dissociationof at least 45° C.
 2. (canceled)
 3. The compound of claim 1, wherein theoligonucleotide analog is composed of morpholino subunits linked byphosphorous-containing intersubunit linkages that join a morpholinonitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit.4. The compound of claim 3, wherein the morpholino subunits are joinedby phosphorodiamidate linkages in accordance with the structure:

where Y₁═O, Z=O, Pj is a purine or pyrimidine base-pairing moietyeffective to bind, by base-specific hydrogen bonding, to a base in apolynucleotide, and X is alkyl, alkoxy, thioalkoxy, or alkyl amino. 5.The compound of claim 3, in which at least 2 and no more than half ofthe total number of intersubunit linkages are positively charged atphysiological pH.
 6. The composition of claim 5, wherein said morpholinosubunits are linked by phosphorodiamidate linkages, in accordance withthe structure:

where Y₁═O, Z=O, Pj is a purine or pyrimidine base-pairing moietyeffective to bind, by base-specific hydrogen bonding, to a base in apolynucleotide, and X for the uncharged linkages is alkyl, alkoxy,thioalkoxy, or an alkyl amino of the form wherein NR₂, where each R isindependently hydrogen or methyl, and for the positively chargedlinkages, X is 1-piperazine.
 7. The compound of claim 1, wherein theoligonucleotide analog hybridizes to a sequence selected from the groupconsisting of SEQ ID NOs:1-9.
 8. The compound of claim 1, wherein theviral target region comprises SEQ ID NO:3 or SEQ ID NO:5.
 9. Thecompound of claim 1, wherein the antisense compound has at least 12contiguous bases from one of the sequences selected from the groupconsisting of SEQ ID NOs:10-24.
 10. The compound of claim 1, wherein thetargeting sequence comprises SEQ ID NO:12 or SEQ ID NO:13.
 11. Thecompound of claim 1, wherein the oligonucleotide analog is conjugated toan arginine-rich polypeptide that enhances the uptake of the compoundinto host cells.
 12. The compound of claim 10, wherein the arginine-richpolypeptide is selected from the group consisting of SEQ ID NOs:25-30.